Lentiviruses encode just over a dozen proteins, but how these proteins cooperate to achieve a persistent infection in primates and humans is complex, involving a network of protein interactions and post-translational modifications. It is well appreciated that viral proteins are multifunctional, but separating and describing these functions, along with their corresponding molecular mechanisms, is still a major challenge for the field. To better understand the multifunctional nature of HIV Vif, this project uses a set of systematic and unbiased methodologies to determine which host factors and pathways are engaged by Vif and how these changes impact viral replication. The most critical function of Vif is to promote viral infectivity by neutralizing the APOBEC3 (A3) family of restriction factors, targeting them for degradation by the 26S proteasome. The Vif protein of primate lentiviruses achieves this through host factor hijacking of both the transcription cofactor CBF? as well as the Cullin5-RING ubiquitin E3 ligase (CRL5). Interactions of Vif with CBF? and CRL5 templates its folding and function, exposing residues for specific interactions with A3 family members resulting in polyubiquitination and degradation. Despite over a decade of research in this area, the ubiquitin machinery regulating A3 turnover has not yet been fully described. Furthermore, several studies indicate Vif may play a role in regulating non-degradative A3 inhibition through an uncharacterized mechanism. At the same time, genome wide transcriptome studies in CD4+ T-cells reveal that Vif has the capacity to downregulate CBF? dependent genes, including the A3 family members, but the mechanism of how this is achieved is also not understood. Here, we propose to tackle these critical gaps in Vif biology in three related, yet distinct aims: 1) we will probe the degree to which Vif re-wires host cell innate immunity by ubiquitination and how this feeds back onto transcription control; 2) we will determine how regulation of the Vif E3 activity depends on interactions with co-factors that promote degradative or non-degradative ubiquitin modifications; and 3) we will examine non-degradative mechanisms of A3 inhibition through Vif-mediated cellular reorganization. This project combines state-of-the art methods to interrogate the complement of proteins that are ubiquitinated by Vif; the coenzymes required for its activity; and its non-degradative functions using unique separation-of-function Fab tools. The biological significance of our findings will be validated by viral infectivity studies in conjunction with gene editing of host factors by CRISPR in primary cells. Completion of this project will provide a comprehensive view of how a single machine, namely the Vif E3 ligase, can serve as a nexus by perturbing multiple host pathways to evade the immune system and promote HIV infectivity.